Solid elements are included in electrophoretic and chromatographic systems for a variety of reasons, including their use as sites for the partitioning of solutes and as retaining walls for housing a separation medium. Solid elements may thus take the form of particles. porous or otherwise, capillary tubes, plates to hold a slab, and other configurations. Examples of chromatographic systems using solid elements are affinity chromatography, reversed phase chromatography, ion exchange chromatography size exclusion chromatography and the various forms of electrophoresis including gel electrophoresis, open-tube electrophoresis, isotachophoresis (also referred to as displacement electrophoresis) and isoelectric focusing. In some cases, the solid element plays an active role in the partitioning, and in other cases a passive role. In some cases, the solid element contributes to the bulk flow of solutes through the separation medium through electroendosmosis (also referred to as electroosmotic flow, as it is herein), while in other cases such flow is considered to be an interference.
Each type of separatory system has its own particular utility. Capillary electrophoresis in its various forms, for example, is significant among electrophoretic systems in general due to a number of advantages which it offers. In particular, it is useful in operations where high speed and efficiency are important. This is because its narrow bore columns promote rapid heat dissipation from the column interior to its surroundings. A high voltage may thus be applied without causing excessive Joule heating. Samples having components which separate only under high voltage can thus be partitioned in such tubes. In addition, the small volume of separation medium used in comparison to other electrophoretic systems lends itself to very small sample sizes. The most common types of capillary electrophoresis are gel electrophoresis, isoelectric focusing, isotachophoresis (also referred to as "displacement" electrophoresis) and free zone electrophoresis, with or without electroosmotic bulk flow.
Wall effects are a major consideration in capillary electrophoresis as well as forms of chromatography. In capillary electrophoresis, there is a high ratio of wall surface area to the volume of the separation medium, and high proximity of the wall to the components being partitioned. In some capillary systems, the presence of an electrokinetic potential is relied on to produce an electroosmotic bulk flow as an integral part of the partition mechanism. Other systems rely primarily on electrophoretic mobility. Electroosmotic flow in such systems interferes with the partitioning, and where present it is sought to be suppressed or eliminated. In many separations, a combination of electrophoretic mobility and electroosmotic flow is used at a ratio which is experimentally derived to provide the cleanest and most efficient separation.
The magnitude of the electrokinetic potential in any electrophoretic system, and hence the degree of electroosmotic flow, are dependent on the surface characteristics of the solid element, which is vulnerable to the various materials which come in contact with it during each separation. These include carrier fluids, buffers, partitioning reagents retained with the carrier, and the solutes sought to be separated. Chemical reactions between these substances and the wall of the solid element, as well as entrapment or retention of these substances by the wall, tend to change the electrical characteristics of the wall, thus affecting the electrokinetic potential. These unwanted interactions with the wall also interfere with the partitioning effect which occurs within the bulk of the separation medium. The changes accumulate with repeated runs, resulting in a loss of reproducibility and, in some cases, to a loss of the partitioning effect itself. The loss of reproducibility is a particularly serious problem in automated systems where a series of samples are injected and partitioned automatically in sequence.
It has now been discovered that an electrokinetic potential may be maintained and reproducibility restored in such systems by treatment of the capillary surface with a redox reagent, i.e., a reagent which promotes either reduction or oxidation on species with which it comes into contact. The choice between oxidation and reduction will depend on the effect sought to be achieved, i.e., the surface electrical character sought to be restored.
For instance, we have found that with silica, oxidizing agents decrease the electrokinetic potential whereas reducing agents increase the electrokinetic potential. The effects are accomplished presumably through modification of surface SiOH groups. With electrophoresis, oxidizing agents would be used when the desired driving force is electrophoretic mobility only, and reducing agents would be used when electroendosmotic flow is the desired driving force (or "pump") for the system. The oxidizing or reducing reagent may serve either a restorative function or a maintenance function, depending on whether it is used as a treatment between runs, or added as a system component during the actual separation.
Further advantages and embodiments of the invention will be apparent from the following description.